RNA interference (RNAi) is a natural method of gene silencing in plant and mammalian cells. RNAi provides a mechanism for the sequence specific silencing of genes. RNAi has been adopted by researchers as a tool to investigate gene function and it has potential applications in the clinical arena such as treatment of neurodegenerative diseases, heart diseases, cancer, and other diseases where silencing of a specific gene or genes is desirable.
However, the efficient delivery of RNAi agents has been a major roadblock on the way to establish RNAi as a suitable gene therapy agent in modern medicine, mostly due to inefficient intake of RNAi agents and quick degradation thereof by RNAses present in blood, lymph, CSF, and intercellular space.
Initial work in the electroporation of siRNAs recommends conditions for transfecting cells in vitro have been to recommend pulse length to 100 μs and pulse voltages from 150-900 V (i.e., 150, 300 . . . 900 V) and then directly transferring the transfected cells to a growth medium (siRNA transfection protocol, Ambion, 2008). Bio-Rad, a maker of in vitro electroportation devices, has published recommended conditions between 200 and 300 volts as optimal for transfection followed by transferring to the cell growth medium (BioTechniques Protocol Guide 2009 (p. 19) doi 10.2144/000113012).
In-Vivo methods for electroporation of RNA to patients has also been described in the literature. In vivo two different electroporation procedures are being used in ongoing clinical trials. In the first procedure, DNA is injected (by needle and syringe) followed by insertion of a four-needle electrode array at the site of injection to deliver electrical pulses, and in the second procedure two standard syringes with injection needles are mounted on a movable sled. As the needles are advanced into the muscle tissue, DNA is injected at a predetermined rate. When DNA injection is completed, electrical pulses are delivered via the two injection needles now serving as electrodes. These clinical studies are sponsored separately by Southampton University Hospitals, and Merck.
Three electroporation devices are known to be approved for use in clinical trials; however, none of these devices are presently commercially available (S. Li (ed.), Electroporation Protocols: Preclinical and Clinical Gene Medicine. 497. From Methods in Molecular Biology, Vol. 423. Humana Press 2008). The first system, the Elgen system, consists of a square wave pulse generator, interfacing with a combined injection/electrode device, which injects the DNA during needle insertion and uses of-the-shelf syringes and needles. The output pulses used in human studies so far were set at a constant current of 250 mA, corresponding to about 60-70 V. The second system, the MedPulser DNA Delivery System (DDS) made by Inovio Biomedical Corporation consists of a pulse generator and a reusable applicator with a disposable tip containing a four-needle array electrode. The MedPulser DDS delivers two unipolar pulses of 60 ms at 106 V, with a frequency of 4 Hz. Typically, DNA vaccine is injected intramuscularly, followed by insertion of the electrode array encompassing the injection site and subsequent pulse delivery. The third system, also made by MedPulser, is the DNA Electroporation Therapy System, and also supplied by Inovio Biomedical Corporation, is similar to the MedPulser DDS. However, it uses a six-needle electrode array, with the needles either integrated into the applicator or contained in a disposable tip (needle length up to 3 cm; electrode distance, 8.6 mm). This system delivers six bipolar, rotating pulses of 100 μs each at 1,130 V, with a frequency of 4 Hz.
So far there does not appear to be any current human studies directed to delivery of RNA molecules subsequent to electroporation. Further the RNA protocol methods are directed to use of relative higher voltages to achieve transfection of these molecules. The present invention overcomes several limitations of the art by providing more efficient delivery of RNAs that does not require the high voltages presently used, which may require sedation during the electroporation process. Further, current electroporation procedures do not provide for chronic delivery of RNA agents. As discussed previously, current delivery protocols couple the nucleic acid delivery with providing the siRNA.
Other delivery methods also have their drawbacks. For example, viral delivery is unproven as an effective in vivo delivery mechanism in humans, and is not approved by the FDA. Lipofection entails administration of extraneous compounds to the patient in addition to the therapeutic agent itself. It is also not approved by the FDA.
Accordingly, new methods of efficient delivery of RNA to the patients are needed.